A Single-Vesicle Electroanalytical Approach Uncovers Deficits of Vesicular Acetylcholine Content in Alzheimer's Disease.

The cholinergic system is essential for cognitive functions including learning, memory, and attention, with its dysfunction serving as a hallmark of neurodegenerative diseases including Alzheimer's disease (AD). However, how acetylcholine (ACh) is stored within individual synaptic vesicles, an essential compartment of cholinergic transmission, remains poorly understood, limiting insight into the nanoscale mechanisms underlying cognitive decline. Here, we present a high-throughput analytical strategy, molecular-empowered vesicle cytometry (MEVC), which employs calix[4]arene-facilitated ion transfer at the interface between two immiscible electrolyte solutions (ITIES) to quantify ACh stored in single vesicles. Validation using ACh-loaded artificial vesicles confirms its robustness and accuracy in quantifying ACh content at the single-vesicle level. Applying MEVC to brain-derived cholinergic vesicles reveals substantial depletion of vesicular ACh storage across multiple regions in an AD mouse model, including the basal forebrain, hippocampus, and cortex, alongside pronounced regional heterogeneity. These findings uncover previously inaccessible alterations in cholinergic neurotransmission and suggest that enhancing ACh synthesis and vesicular packaging may help preserve cognitive function in aging-related diseases. This approach not only paves a new path for sensitive quantification of nonredox-active neurotransmitters at the single-vesicle level, but also holds promise for advancing early diagnostics and therapeutic strategies for neurodegeneration.